Genetic combination, nannochloropsis engineering strain and application thereof in preparation of 24-methylenecholesterol

Abstract

The present application relates to the technical field of biotechnology, in particular to a gene combination, a microalgae engineering strain and its application in preparing 24-methylenecholesterol. Through further inactivating SMT2 gene on the basis of marine microalgae DWF1 gene mutant strain, high-efficiency accumulation of 24-methylenecholesterol is realized, and the proportion of 24-methylenecholesterol in total sterol is more than 96%. The DWF1 and SMT2 gene mutant strain provided by the present application significantly reduces the content of cholesterols and other by-product sterols, and improves the purity of algal 24-methylenecholesterol. The MEM32 engineering strain constructed by the present application is suitable for conventional microalgae culture system, and is convenient for subsequent scale-up culture and large-scale preparation of algal 24-methylenecholesterol. The present application provides a biological preparation material for high-purity algal 24-methylenecholesterol. The prepared algal 24-methylenecholesterol has the effects of antioxidant, anti-aging, life extension, improved exercise capacity and improved oxidative stress tolerance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to gene combinations, engineered strains of *Microcystis aeruginosa*, and their application in the preparation of 24-methylene cholesterol. Background Technology

[0002] Marine bioactive substances have broad application prospects in medicine, functional foods, and cosmetics. However, most natural active ingredients are present in extremely low concentrations in organisms, and their scarcity, complex extraction processes, and high preparation costs are due to factors such as marine biological resources, harvesting costs, geographical distribution, and ecological protection. This severely restricts the industrial development and in-depth research of related high-value compounds. For example, oyster sterol, a natural sterol compound with a unique side-chain structure, may show significant advantages in potential effects such as antioxidation, anti-inflammation, lipid-lowering, and neuroprotection. However, its natural sources are mainly limited to certain marine invertebrates (such as oysters and sponges), with extremely low concentrations and high difficulty in separation and purification, which can easily cause environmental pollution and makes it difficult to meet the needs of systematic functional evaluation and large-scale application.

[0003] The development of synthetic biology has provided a novel approach to addressing the scarcity of marine natural product sources. Constructing heterologous synthesis systems to achieve efficient and targeted synthesis of target compounds in scalable chassis microorganisms has become an important direction for the sustainable utilization of marine pharmaceutical resources. *Nannochloropsis* spp. is an important marine industrial microalga with significant advantages such as rapid growth, short cultivation cycles, increasingly mature genetic manipulation, and suitability for large-scale outdoor seawater cultivation. Furthermore, it possesses a naturally complete sterol synthesis pathway, making it an excellent chassis cell for the heterologous or enhanced expression of marine-derived sterol compounds. Therefore, there is an urgent need to provide technical solutions for constructing synthetic biology systems based on *Nannochloropsis* to achieve efficient and targeted production of specific sterols from sunlight-driven CO2 and clean seawater, thereby overcoming the resource and cost bottlenecks of natural extraction methods. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide gene combinations, engineered strains of *Microcystis aeruginosa* and their application in the preparation of 24-methylene cholesterol. The present invention provides a method for constructing marine *Microcystis aeruginosa* enriched with oyster sterol 24-methylene cholesterol, the development of bioproducts and their application in anti-aging, life extension, improvement of exercise capacity and enhancement of oxidative stress tolerance.

[0005] This invention provides the application of a combination of inhibiting and / or knocking out genes in regulating the production of at least one of cholesterol, sterols, 24-methylene cholesterol, sterol, isoflavone, fucosterol and total sterols by *Microcystis aeruginosa*, said gene combination including the DWF1 gene and the SMT2 gene.

[0006] In some embodiments, the DWF1 gene has a nucleotide sequence as shown in SEQ ID NO:1, and the SMT2 gene has a nucleotide sequence as shown in SEQ ID NO:1.

[0007] The present invention provides sgRNAs for knocking out the DWF1 and SMT2 genes, comprising a first sgRNA with a nucleotide sequence as shown in SEQ ID NO:3 and / or a second sgRNA with a nucleotide sequence as shown in SEQ ID NO:4.

[0008] This invention provides vector plasmids for knocking out the DWF1 and SMT2 genes, including a first vector plasmid and / or a second vector plasmid:

[0009] The first vector plasmid comprises a nucleic acid molecule encoding the Cas9 protein and the first sgRNA as described in claim 3;

[0010] The second vector plasmid comprises a nucleic acid molecule encoding the Cas9 protein and the second sgRNA as described in claim 3.

[0011] Using the aforementioned sgRNA or vector plasmids, the knockout efficiency of DWF1 and SMT2 genes is higher and the off-target rate is lower, thus obtaining more accurate experimental results.

[0012] This invention provides an engineered strain of *Microcystis aeruginosa*, which is obtained by transforming the vector plasmid into wild-type *Microcystis aeruginosa* to obtain a dwf1 / smt2 double knockout mutant; or

[0013] Wild-type *Microcystis globulus* was used to obtain the dwf1 / smt2 double knockout mutant through artificial random mutagenesis.

[0014] This invention provides a method for preparing the aforementioned engineered strain of *Microcystis aeruginosa*, which involves transforming the vector plasmid into wild-type *Microcystis aeruginosa*; or

[0015] Wild-type Micrococcus microcarpa was used to obtain the dwf1 / smt2 double knockout mutant through artificial random mutagenesis.

[0016] The method for constructing the MEM32 engineered strain provided by the present invention also includes artificial mutagenesis of marine microglobulus, screening to obtain double mutant strains of DWF1 and SMT2 genes, and achieving over-synthesis of oyster sterols under the premise that the mutant strains produce beneficial effects or are unaffected or only slightly affected.

[0017] This invention provides the application of the sgRNA, the vector plasmid, the engineered strain of *Microcystis aeruginosa*, and / or the engineered strain of *Microcystis aeruginosa* prepared by the method described above in the preparation of 24-methylene cholesterol.

[0018] The present invention provides a method for preparing 24-methylene cholesterol, comprising: culturing the *Microcystis aeruginosa* engineered strain and / or the *Microcystis aeruginosa* engineered strain obtained by the preparation method described above to obtain 24-methylene cholesterol.

[0019] In some embodiments, the culture medium is F / 2 medium, the culture temperature is 25 ℃, the light intensity is 50 μmol·m⁻²·s⁻¹, and the light-dark cycle is 14h light: 10h dark.

[0020] This invention provides the application of 24-methylene cholesterol prepared by the method described above in the preparation of antioxidant and / or anti-aging products.

[0021] Antioxidant and / or anti-aging products, including excipients and 24-methylene cholesterol prepared by the method described above.

[0022] Methods of anti-oxidation and / or anti-aging include administering the product.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) By further inactivating the SMT2 gene on the basis of the marine microcystis DWF1 gene mutant strain (24-methylene cholesterol accounts for about 50% of the total sterols), the efficient accumulation of 24-methylene cholesterol was achieved, which accounted for more than 96% of the total sterols.

[0025] (2) The DWF1 and SMT2 gene mutant strains provided by the present invention significantly reduced the content of cholesterol and other by-product sterols and improved the purity of algal 24-methylene cholesterol.

[0026] (3) The MEM32 engineered strain constructed in this invention is suitable for conventional microalgae culture systems, which facilitates subsequent scale-up culture and large-scale preparation of algal-derived 24-methylene cholesterol, and provides biomaterials for the preparation of high-purity algal-derived 24-methylene cholesterol.

[0027] (5) The present invention has been verified by the Caenorhabditis elegans model. The algal 24-methylene cholesterol obtained by the present invention has the effects of anti-oxidation, anti-aging, prolonging life, improving exercise ability and improving oxidative stress tolerance. Attached Figure Description

[0028] Figure 1 Sequencing results of SMT2 and DWF1 genes in the marine microcystis MEM32 strain are shown.

[0029] Figure 2The sterol profiles of wild-type, MEM31, and MEM32 strains of marine micronaria are shown. Among them, WT is wild-type micronaria; MEM31 is a dwf1 mutant strain; and MEM32 is a dwf1 / smt2 mutant strain.

[0030] Figure 3 This paper presents the efficacy evaluation of marine microalgae MEM32 steroidal biological products. Figure A shows the lifespan detection results of *C. elegans*; Figure B shows the head twitching frequency detection results of *C. elegans*; Figure C shows the pharyngeal pump frequency detection results of *C. elegans*; Figure D shows the CAT activity detection results of *C. elegans*. In the figures, a, b, and c refer to different treatment groups with significant differences (P < 0.05); CHOL refers to the cholesterol treatment group; WT refers to the wild-type marine microalgae sterol extract; MEM32 refers to the marine microalgae MEM32 strain sterol extract; and 24-MC refers to the pure 24-MC (concentration > 99.85%) obtained by high-performance liquid chromatography purification using MEM32 as raw material.

[0031] Figure 4 The effect of MEM32-derived 24-MC on superoxide dismutase activity in Caenorhabditis elegans was shown, with the cholesterol-treated group as the control. Data are expressed as mean ± standard deviation (n=3), and there was no significant difference between the two groups (*p >0.05).

[0032] Figure 5 The effect of MEM32-derived 24-MC on the total antioxidant capacity of Caenorhabditis elegans was shown, with the cholesterol-treated group as the control. Data are expressed as mean ± standard deviation (n=3), and there was no significant difference between the two groups (*p>0.05).

[0033] Figure 6 The effect of MEM32-derived 24-MC on the total catalase activity of Caenorhabditis elegans was shown, with the cholesterol-treated group as the control. Data are expressed as mean ± standard deviation (n=3), and *p<0.05 indicates a significant difference compared with the control group.

[0034] Figure 7 The effect of MEM32-derived 24-MC on malondialdehyde activity in Caenorhabditis elegans was shown, with the cholesterol-treated group as the control. Data are expressed as mean ± standard deviation (n=3), and there was no significant difference between the two groups. Detailed Implementation

[0035] This invention provides gene combinations, engineered strains of *Microcystis aeruginosa*, and their application in the preparation of 24-methylene cholesterol. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0036] In the sterol metabolic network of *Microcystis globulus*, cholesterol-rich sterols and various steroid metabolites are highly correlated with the side-chain structure of oysterol, suggesting that algal synthesis of oysterol can be achieved through heterologous introduction of sterol side-chain modifying enzymes or reprogramming of endogenous pathways. Our previous research team discovered that knocking out the native DWF1 sterol in *Microcystis globulus* significantly increased oysterol yield (from zero to over 50% of total sterols). This study further explores targeted knockout of SMT2 (responsible for C-24 methylation) to further enhance the synthesis and accumulation of oysterol in the *Microcystis globulus* chassis. Based on this, the potential application value of algal oysterol in anti-aging and neuroprotection was explored. Developing an efficient oysterol synthesis system based on *Microcystis globulus* not only provides a feasible technical path to overcome the scarcity and extraction difficulties of marine bioactive sterols, but also lays the foundation for promoting their application in functional foods, cosmetics, and innovative drugs.

[0037] To achieve the above objectives, the present invention adopts the following technical solution:

[0038] This invention provides a MEM32 engineered strain of *Microcystis globulus* rich in 24-methylene cholesterol, which is obtained by further inactivating the SMT2 gene based on the *Microcystis globulus* DWF1 gene mutant strain.

[0039] The DWF1 gene is the gene encoding Δ24-sterol reductase; the SMT2 gene is the gene encoding sterol methyltransferase 2, which participates in the methylation reaction at the C-24 position of the sterol side chain.

[0040] In the MEM32 engineered strain, 24-methylene cholesterol accounts for more than 96% of the total sterols.

[0041] The present invention also provides a method for constructing the MEM32 engineered strain. The method uses the CRISPR / Cas9 gene editing system to perform site-specific editing of the SMT2 gene in the marine microcystis DWF1 gene mutant strain to inactivate it, thereby obtaining the MEM32 engineered strain enriched with 24-methylene cholesterol.

[0042] The present invention also provides a method for constructing the MEM32 engineered strain, which further includes artificially inducing mutagenesis of marine microglobulus to screen and obtain double mutant strains of DWF1 and SMT2 genes, and achieving the over-synthesis of oysterols under the premise that the growth of the mutant strains is not affected or produces beneficial effects.

[0043] The present invention further provides the application of the MEM32 engineered strain in the preparation of algal-derived 24-methylene cholesterol.

[0044] The present invention further provides the application of algal-derived 24-methylene cholesterol from the MEM32 engineered strain in anti-aging effects.

[0045] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0046] Example 1: Construction of the marine microcystis MEM32 strain

[0047] This embodiment provides a method for constructing a marine microcystis strain that produces high levels of oyster sterol.

[0048] Based on the DWF1 gene mutant strain MEM31 of *Microcystis globulus* (preparation method detailed in CN114058515A), the SMT2 gene was inactivated by site-specific editing using the CRISPR / Cas9 gene editing system. The SMT2 gene encodes sterol methyltransferase 2 and participates in the methylation reaction at position C-24 of the sterol side chain. The DWF1 gene encodes Δ24-sterol reductase, and its cDNA sequence is shown in SEQ ID NO:1. The sgRNA targeting sequence for DWF1 gene editing is shown in SEQ ID NO:3. The cDNA sequence of the SMT2 gene is shown in SEQ ID NO:2, and the sgRNA targeting sequence for SMT2 gene editing is shown in SEQ ID NO:4.

[0049] The cDNA sequence of the DWF1 gene is shown below:

[0050]

[0051] The SMT2 gene cDNA sequence is shown below:

[0052]

[0053] sgRNA targeting sequence for DWF1 gene editing:

[0054] 5'-GGGCTTCCCTTTGCCTTGG-3' (SEQ ID NO:3)

[0055] sgRNA targeting sequence for SMT2 gene editing:

[0056] 5'-CCATTGTCGTTTTCCGCGTTCGA-3' (SEQ ID NO:4)

[0057] Electroporation transformation was performed according to Wang et al. (Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR / Cas9). Gene knockout was performed according to Lu et al. (Role of an ancient light-harvesting protein of PSI in light absorption and photoprotection). The transformed algal culture was plated on F / 2 plates containing bleomycin. Single colonies were selected and cultured in liquid medium at 25°C and a light intensity of 50 μmol·photons·m⁻¹. -2 ·s -1 Incubation period: 21 days.

[0058] Specifically, the CRISPR / Cas9 vector used for SMT2 knockout was electroporated into the MEM31 algal strain, and candidate engineered algal strains were obtained through screening. PCR amplification and Sanger sequencing were performed on the candidate algal strains for verification. The results showed that the SMT2 gene had insertion or deletion mutations (only partial results are shown here), indicating that the SMT2 gene was successfully edited and inactivated. A total of 8 mutant strains were obtained, named MEM32-1 to MEM32-8. The above gene knockout method is only used to illustrate the construction concept of the MEM32 strain, but is not limited to gene editing methods; any method that can achieve mutations in the DWF1 and SMT2 genes (such as random mutagenesis) is included within the scope of protection of this invention.

[0059] The MEM32 engineered strain can be cultured using an F / 2 culture system. Preferably, the culture temperature is 25 ℃, the light intensity is 50 μmol·m⁻²·s⁻¹, and the light-dark cycle is 14 h:10 h. These culture conditions are typical for microalgae culture and are used to illustrate the culturability and feasibility of the MEM32 engineered strain.

[0060] In this embodiment, Smt1 mutant, Smt2 mutant, and smt1 / dwf1 mutant were further constructed. The cDNA sequence of the SMT1 gene is shown in SEQ ID NO:5, and the sgRNA targeting sequence for SMT1 gene editing is shown in SEQ ID NO:6.

[0061] The SMT1 gene cDNA sequence is shown below:

[0062]

[0063] sgRNA targeting sequence for SMT1 gene editing:

[0064] 5'-GAGGGGGAATCGTAGTGTGGTGG-3' (SEQ ID NO: 6)

[0065] Smt1 mutant: Based on wild-type marine micrococcus, the SMT1 gene was inactivated by site-specific editing using the CRISPR / Cas9 gene editing system.

[0066] Smt2 mutant: Based on wild-type marine micrococcus, the SMT2 gene was inactivated by site-specific editing using the CRISPR / Cas9 gene editing system.

[0067] smt1 / dwf1 mutant: Based on the DWF1 gene mutant strain MEM31 of marine microcystis, the SMT1 gene was edited at specific sites using the CRISPR / Cas9 gene editing system to inactivate the SMT1 gene.

[0068] Example 2: Determination of sterol composition in the MEM32 strain

[0069] This example illustrates that the MEM32 engineered strain can significantly enrich 24-methylene cholesterol.

[0070] Wild-type marine microcystis, MEM31 and MEM32 samples were collected and their sterol composition was analyzed by GC-MS (only data from one engineered strain are used here for illustration).

[0071] GC-MS analysis parameters: flow rate 1 mL / min, initial temperature 170°C, hold for 1 min, then increase to 280°C at 20°C / min and hold for at least 15 min, ion source temperature 150°C, injection volume 1 μL, column oven temperature 170°C.

[0072] The test results showed that cholesterol was the main sterol component in wild-type marine microcystis; 24-methylene cholesterol accounted for about 64% of the total sterols in MEM31; and 24-methylene cholesterol accounted for more than 96% of the total sterols in engineered strain MEM32, while cholesterol, isofostellarol and fucosterol were not detected.

[0073] The specific results are shown in Table 1 below:

[0074] Table 1

[0075]

[0076] The above results indicate that the dual inactivation of the DWF1 / SMT2 genes altered the metabolic flow of marine microphytoid algae sterols, significantly increasing the proportion of 24-methylene cholesterol in total sterols.

[0077] Further comparison of 24-methylene cholesterol content in smt1 mutant, smt2 mutant and smt1 / dwf1 mutant lines revealed that the 24MC content was not increased in the smt1 mutant (24MC content was 0%), while the 24MC content in the smt1 / dwf1 double knockout line was approximately 48%, which was lower than that in the MEM31 and MEM32 knockout lines.

[0078] Application of Algal Source 24-Methylene Cholesterol in Evaluation of Antioxidant and Anti-aging Efficacy of Online Insecticidal Cells

[0079] 1. The MEM32 strain obtained in Example 1 was cultured, the algal cells were collected, and the sterol components were extracted to obtain an algal bioproduct (MEM32) rich in 24-methylene cholesterol. The algal bioproduct (MEM32) rich in 24-methylene cholesterol was purified using a high-performance liquid chromatography (HPLC) fractionation device to obtain pure 24-MC (concentration >99.85%). Similarly, the wild-type strain of *Microcystis aeruginosa* was cultured, the algal cells were collected, and the sterol components were extracted to obtain an algal bioproduct (WT).

[0080] The aforementioned algal-derived biological products were added to *E. coli* OP50 bacterial suspension to feed *C. elegans*. Wild-type *Microcystis globosum* extract (WT), a cholesterol-fed group (CHOL), and 24-MC purified from MEM32 were used as controls. The lifespan, motility, and oxidative stress tolerance of the nematodes were measured. The lifespan of the nematodes was determined using conventional lifespan testing methods; motility was evaluated by observing the nematode movement; and oxidative stress tolerance was assessed by assessing the survival of the nematodes under oxidative stress conditions. The lifespan of the nematodes was determined using conventional lifespan testing methods, and the average lifespan of each treatment group and the lifespan extension rate relative to the CHOL control group were calculated. Motility was evaluated by detecting the nematode head twitching frequency and pharyngeal pump frequency. Oxidative stress-related indicators were evaluated by detecting CAT activity. One-way ANOVA combined with Tukey's multiple comparison method was used to analyze differences between different groups. Figure 3 In Figures B and C, letters a, b, and c represent the results of the significance analysis of differences between groups. Different letters indicate significant differences between groups (P < 0.05), while the same letter indicates no significant differences between groups (P ≥ 0.05). Figure 3 In the D-plot, * indicates a significant difference between groups (P < 0.05).

[0081] The specific dosages for different treatment groups are shown in Table 2 below:

[0082] Table 2

[0083]

[0084] The results are as follows Figure 3 As shown in the figure. Compared with the CHOL control group, the average lifespan of nematodes in the WT treatment group increased from 13.28 days to 14.48 days, with a lifespan extension rate of 9.04%; the average lifespan of nematodes in the MEM32 treatment group further increased to 15.78 days, with a lifespan extension rate of 18.83%; and the average lifespan of nematodes in the purified 24-MC treatment group increased to 16.25 days, with a lifespan extension rate of 22.41%, further enhancing the lifespan extension effect. Figure 3 A). Meanwhile, compared to the WT group, the MEM32 treatment group showed an increased head-shaking frequency in nematodes ( Figure 3 B); Compared with the CHOL control group, the MEM32 treatment group showed an increased nematode pharyngeal pump frequency (B); Figure 3 C), CAT activity is enhanced under oxidative stress ( Figure 3 D). The above results indicate that the algal sterol components derived from the MEM32 engineered strain have a more significant anti-aging effect than wild-type Micrococcus microcarpa, and the purified 24-MC treatment group showed similar or stronger effects, indicating that the MEM32 bioproduct has anti-aging activity.

[0085] The above results indicate that oysterol can effectively enhance the motility and antioxidant capacity of Caenorhabditis elegans and delay aging; while MEM32 is an important source of oysterol and can be used to develop bioproducts with health benefits.

[0086] 2. Using the cholesterol-treated group as a control, the effect of MEM32-derived 24-MC on the activity of superoxide dismutase in nematodes was determined.

[0087] The superoxide dismutase (SOD) activity of nematodes in each experimental group was measured and statistically analyzed. The results showed that 24-MC treatment had no effect on SOD levels in nematodes. Figure 4 ).

[0088] 3. Using the cholesterol-treated group as a control, the effect of MEM32-derived 24-MC on the total antioxidant capacity of nematodes was determined.

[0089] The total antioxidant capacity of nematodes in each experimental group was measured and statistically analyzed. The results showed that 24-MC treatment had no effect on the T-AOC level in nematodes (…). Figure 5 ).

[0090] 4. Using the cholesterol-treated group as a control, the effect of MEM32-derived 24-MC on the total catalase activity of nematodes was determined.

[0091] The total catalase activity of nematodes in each experimental group was measured and statistically analyzed. The results showed that, compared with the cholesterol control group, the CAT activity in *C. elegans* treated with 24-MC was significantly upregulated, reaching 3.1 times that of the control group. Figure 6 ).

[0092] Previous studies have shown that CAT is a core antioxidant enzyme in organisms that scavenges hydrogen peroxide. It can effectively degrade reactive oxygen species (ROS) accumulated during aging, alleviate cell and tissue damage caused by oxidative stress, and enhance antioxidant enzyme activity. Therefore, 24-MC treatment can significantly activate the antioxidant defense system of nematodes, enhance the body's ability to scavenge ROS, and reduce the accumulation of oxidative damage, thereby alleviating the oxidative aging process of nematodes.

[0093] In summary, algal-derived 24-MC can significantly enhance the activity of CAT antioxidant enzymes in nematodes, thereby increasing their tolerance to oxidative stress. 24-MC can regulate the aging process of nematodes by activating their endogenous antioxidant system and reducing the accumulation of aging-related oxidative damage, ultimately achieving a biological effect of extending lifespan.

[0094] 5. Using the cholesterol-treated group as a control, the effect of MEM32-derived 24-MC on malondialdehyde activity in nematodes was determined.

[0095] The malondialdehyde (MDA) activity of nematodes in each experimental group was measured and statistically analyzed. The results showed that 24-MC treatment had no effect on the MDA level in nematodes. Figure 7 ).

[0096] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a combination of inhibited and / or knocked-out genes in regulating the production of at least one of cholesterol, sterol, 24-methylene cholesterol, sterol, isoflavone, fucosterol and total sterols by *Microcystis aeruginosa*, said gene combination including the DWF1 gene and the SMT2 gene.

2. The application according to claim 1, characterized in that, The DWF1 gene has the nucleotide sequence shown in SEQ ID NO:1, and the SMT2 gene has the nucleotide sequence shown in SEQ ID NO:

1.

3. A method for knocking out the sgRNA of the DWF1 and SMT2 genes, characterized in that... Includes a first sgRNA with the nucleotide sequence shown in SEQ ID NO:3 and / or a second sgRNA with the nucleotide sequence shown in SEQ ID NO:

4.

4. A vector plasmid that knocks out the DWF1 and SMT2 genes, characterized in that, Including the first vector plasmid and / or the second vector plasmid: The first vector plasmid comprises a nucleic acid molecule encoding the Cas9 protein and the first sgRNA as described in claim 3; The second vector plasmid comprises a nucleic acid molecule encoding the Cas9 protein and the second sgRNA as described in claim 3.

5. An engineered strain of *Microcystis aeruginosa*, characterized in that, It involves transforming the vector plasmid described in claim 4 into wild-type *Microcystis globulus* to obtain the dwf1 / smt2 double knockout mutant; or Wild-type Micrococcus microcarpa was used to obtain the dwf1 / smt2 double knockout mutant through artificial random mutagenesis.

6. The method for preparing the *Microcystis aeruginosa* engineered strain according to claim 5, characterized in that, It involves transforming the vector plasmid of claim 4 into wild-type Micrococcus microcarpa; or Wild-type Micrococcus microcarpa was used to obtain the dwf1 / smt2 double knockout mutant through artificial random mutagenesis.

7. The use of the sgRNA of claim 3, the vector plasmid of claim 4, the engineered strain of *Microcystis aeruginosa* of claim 5, and / or the engineered strain of *Microcystis aeruginosa* prepared by the method of claim 6 in the preparation of 24-methylene cholesterol. A method for preparing 8,24-methylene cholesterol, characterized in that, include: 24-methylene cholesterol was obtained by culturing the *Microcystis aeruginosa* engineered strain as described in claim 5 and / or the *Microcystis aeruginosa* engineered strain prepared by the preparation method described in claim 6.

9. The use of 24-methylene cholesterol prepared by the method of claim 8 in the preparation of antioxidant and / or anti-aging products.

10. An antioxidant and / or anti-aging product, characterized in that, Includes excipients and 24-methylene cholesterol prepared by the method described in claim 8.

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